Method and system for myocardial infarction repair

ABSTRACT

An implantable system is provided that includes: a cell repopulation source comprising genetic material, undifferentiated contractile cells, or a combination thereof capable of forming new contractile tissue in and/or near an infarct zone of a patient&#39;s myocardium; and an electrical stimulation device for electrically stimulating the new contractile tissue in and/or near the infarct zone of the patient&#39;s myocardium.

CROSS-REFERENCE TO RELATED TECHNOLOGY

This application is a continuation of Ser. No. 09/145,743, now U.S. Pat.No. 6,151,525 which is a continuation of U.S. application Ser. No.09/145,743, filed Sep. 2, 1998.

This application claims priority from a provisional patent applicationfiled on Nov. 7, 1997 entitled “Method and Apparatus for MyocardialInfarct Repair” and assigned Provisional Serial No. 60/064,703, which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods and implantable systems toreverse damage to heart muscle following myocardial infarction.Specifically, this involves the repopulation of the damaged myocardiumwith undifferentiated contractile cells, which may be formed in situthrough the use of genetic engineering techniques, and augmentation withelectrical stimulation.

BACKGROUND OF THE INVENTION

Coronary Artery Disease (CAD) affects 1.5 million people in the USAannually. About 10% of these patients die within the first year andabout 900,000 suffer from acute myocardial infarction. During CAD,formation of plaques under the endothelial tissue narrows the lumen ofthe coronary artery and increases its resistance to blood flow, therebyreducing the O₂ supply. Injury to the myocardium (i.e., the middle andthickest layer of the heart wall, composed of cardiac muscle) fed by thecoronary artery begins to become irreversible within 0.5-1.5 hours andis complete after 6-12 hours, resulting in a condition called acutemyocardial infarction (AMI) or simply myocardial infarction (MI).

Myocardial infarction is a condition of irreversible necrosis of heartmuscle that results from prolonged ischemia. Damaged regions of themyocardium are infiltrated with noncontracting scavenger cells andultimately are replaced with scar tissue. This fibrous scar does notsignificantly contribute to the contraction of the heart and can, infact, create electrical abnormalities.

Those who survive AMI have an 4-6 times higher risk of developing heartfailure. Current and proposed treatments for those who survive AMI focuson pharmacological approaches and surgical intervention. For example,angioplasty, with and without stents, is a well known technique forreducing stenosis. Most treatments are designed to achieve reperfusionand minimize ventricular damage. However, none of the current orproposed therapies address myocardial necrosis (i.e., degradation anddeath of the cells of the heart muscle). Because cardiac cells do notdivide to repopulate the damaged region, this region will fill withconnective tissue produced by invading fibroblasts. Fibroblasts produceextracellular matrix components of which collagen is the most abundant.Neither the fibroblasts themselves nor the connective tissue they formare contractile. Thus, molecular and cellular cardiomyoplasty researchhas evolved to directly address myocardial necrosis.

Cellular cardiomyoplasty involves transplanting cells, rather thanorgans, into the damaged myocardium with the goal of restoring itscontractile function. Research in the area of cellular cardiomyoplastyis reviewed in Cellular Cardiomyoplasty: Myocardial Repair with CellImplantation, ed. Kao and Chiu, Landes Bioscience (1997), particularlyChapters 5 and 8. For example, Koh et al., J. Clinical Invest., 96,2034-2042 (1995), grafted cells from AT-1 cardiac tumor cell line tocanines, but found uncontrolled growth. Robinson et al., CellTransplantation, 5, 77-91 (1996),grafted cells from C₂C₁₂ skeletalmuscle cell line to mouse ventricles. Although these approaches producedintriguing research studies, cells from established cell lines aretypically rejected from the human recipient. Li et al., Annals ofThoracic Surgery, 62, 654-661 (1996), delivered fetal cardiomyocytes toadult mouse hearts. They found improved systolic pressures and noticedthat the presence of these cells prevented remodeling after theinfarction. Although their results showed the efficacy of transplantedcell technology, this approach would not likely be effective in clinicalmedicine since the syngeneic fetal cardiac tissue will not be availablefor human patients. Chiu et al., Ann. Thorac. Surg., 60, 12-18 (1995)performed direct injection of cultured skeletal myoblasts to canineventricles and found that well developed muscle tissue could be seen.This method, however, is highly invasive, which compromises itsfeasibility on human MI patients.

Molecular cardiomyoplasty has developed because fibroblasts can begenetically manipulated. That is, because fibroblasts, which are notterminally differentiated, arise from the same embryonic cell type asskeletal muscle, their phenotype can be modified, and possibly convertedinto skeletal muscle satellite cells. This can be done by turning onmembers of a gene family (myogenic determination genes or “MDGS”)specific for skeletal muscle. A genetically engineered adeno-viruscarrying the myogenin gene can be delivered to the MI zone by directinjection. The virus penetrates the cell membrane and uses the cell'sown machinery to produce the myogenin protein. Introduction of themyogenin protein into a cell turns on the expression of the myogeningene, which is a skeletal muscle gene, and which, in turn, switches onthe other members of the MDGS and can transform the fibroblast into askeletal myoblast. To achieve this gene cascade in a fibroblast,replication deficient adenovirus carrying the myogenin gene can be usedto deliver the exogenous gene into the host cells. Once the virusinfects the fibroblast, the myogenin protein produced from the viralgenes turns on the endogenous genes, starting the cascade effect, andconverting the fibroblast into a myoblast. Without a nuclear envelope,the virus gets degraded, but the cell's own genes maintain the cell'sphenotype as a skeletal muscle cell.

This concept has been well-developed in vitro. For example, Tam et al.,J. Thoracic and Cardiovascular Surgery, 918-924 (1995), used MyoDexpressing retrovirus in vitro for fibroblast to myoblast conversion.However, its viability has not been demonstrated in vivo. For example,Klug et al., J. Amer. Physiol. Society, 1913-1921 (1995), used SV40 invivo and succeeded in replicating the nucleus and DNA, but not thecardiomyocytes themselves. Also, Leor et al., J. Molecular and CellularCardiology, 28, 2057-2067 (1996), reported the in situ generation of newcontractile tissue using gene delivery techniques.

Thus, there is a need for an effective system and the method for lessinvasive delivery of a source of repopulating agents, such as cells orvectors, to the location of the infarct zone of the myocardium.

Many of the following lists of patents and nonpatent documents discloseinformation related to molecular and cellular cardiomyoplastytechniques. Others are directed to background information on myocardialinfarction, for example.

TABLE 1a Patents Patent No. Inventor(s) 4,379,459 Stein 4,411,268 Cox4,476,868 Thompson 4,556,063 Thompson et al. 4,821,723 Baker et al.5,030,204 Badger et al. 5,060,660 Gambale et al. 5,069,680 Grandjean5,104,393 Isner et al. 5,131,388 Pless 5,144,949 Olson 5,158,078 Bennettet al. 5,205,810 Guiraudon et al. 5,207,218 Carpentier et al. 5,312,453Shelton et al. 5,314,430 Bardy 5,354,316 Keimel 5,510,077 (Dinh et al.)5,545,186 Olson et al. 5,658,237 Francishelli 5,697,884 Francishelli etal.

TABLE 1b Foreign Patent Documents Document No. Applicant PublicationDate WO 93/04724 Rissman et al. 03/15/93 WO 94/11506 Leiden et al.05/26/94 WO 95/05781 Mulier et al. 03/02/95 WO 97/09088 Elsberry et al.03/13/97

TABLE 1c Nonpatent Documents Acsadi et al, The New Biol., 3, 71-81(1991). Barr et al., Gene Ther., 1, 51-58 (1994). CellularCardiomyoplasty: Myocardial Repair with Cell Implantation, ed. Kao andChiu, Landes Bioscience (1997) Chiu et al., “Cellular Cardiomyoplasty:Myocardiol Regeneration With Satellite Cell Implantation”, Ann. Thorac.Surg., 60, 12-18 (1995). Fletcher et al., “Acute Myocardiol Infarction”,Pathophysiology of Heart Disease, French et al., Circulation, 90,2414-2424 (1994). Gal et al., Lab. Invest., 68, 18-25 (1993). Innis etal. Eds. PCR Strategies, 1995, Academic Press, New York, New York.Johns, J. Clin. Invest., 96, 1152-1158 (1995). Klug et al., J. Amer.Physiol. Society, 1913-1921 (1995). Koh et al., J. Clinical Invest., 96,2034-2042 (1995). Leor et al., J. Molecular and Cellular Cardiology, 28,2057-2067 (1996) Li et al., Annals of Thorasic Surgery, 60, 654-661(1996). Mesri et al., “Expression of Vascular Endothelial Growth FactorFrom a Defective Herpes Simplex Virus Type 1 Amplicon Vector InducesAngiogenesis in Mice”, Department of Medicine, Division ofEndocrinology, Diabetes Research Center, Bronx, New York (Received08/19/94, accepted 11/03/94), 1995, American Heart Association.Molecular Cloning: A Laboratory Manual, 1989 Cold Spring HrborLaboratory Press, Cold Spring Harbor, New York. Murry et al., J. Clin.Invest., 98, 2209-2217 (1196) Olson, “Remington's PharmaceuticalSciences”, a standard reference text in this field. Parmacek et al, J.Biol. Chem., 265, 15970-15976 (1990). Parmacek et al., Mol. Cell. Biol.,12, 1967-1976 (1992). Robinson et al., Cell Transplantation, 5, 77-91(1996). Robinson et al., “Implantation of Skeletal Myoblast-DerivedCells”, Cellular Cardiomyoplasty: Myocardiol Repair with CellImplantation, eds. R. Kao and R. C-J. Chiu, Landes Bioscience (1997).Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York. Symes,“Therapeutic Angiogenesis for Coronary Artery and Peripheral VascularDisease”, LAD, July 1997 (XIX Annual Meeting of ISHR- American Section.Tam et al., J. Thorasic and Cardiovascular Surgery, 918-924 (1995).Taylor et al., “Delivery of Primary Autologous Skeletal Myoblasts intoRabbit Hear by Coronary Infusion: A Potential Approach to MyocardialRepair”, Proceedings of the Association of American Physicians, 109,245-253 (1997). von Recumin et al., Biomaterials, 12, 385-389,“Texturing of Polymer Surfaces at the Cellular Level” (1991). vonRecumin et al., Biomaterials, 13, 1059-1069, “Macrophage Response toMicrotextured Silicone” (1992). von Recumin et al., Journal ofBiomedical Materials Research, 27, 1553- 1557, “Fibroblast Anchorage toMicrotextured Surfaces” (1993). Zibaitis et al., “CellularCardiomyoplasty: Biological Basis, Current Hypothesis and FuturePerspective”, Cellular Cardiomyoplasty: Myocardiol Repair with CellImplantation, eds. R. Kao and R. C-J. Chiu, Landes Bioscience (1997).

All patent and nonpatent documents listed in Table 1 are herebyincorporated by reference herein in their respective entireties. Asthose of ordinary skill in the art will appreciate upon reading theSummary of the Invention, Detailed Description of Preferred Embodiments,and Claims set forth below, many of the systems, devices, and methodsdisclosed in these documents may be modified advantageously by using theteachings of the present invention.

SUMMARY OF THE INVENTION

The present invention provides methods and implantable systems thatreverse the damage to necrotic heart muscle following myocardialinfarction. Specifically, this involves combining a method of supplyinga source of a repopulating agent with a stimulation device. Morespecifically, this involves the repopulation of the damaged myocardiumwith undifferentiated contractile cells and augmentation of the newlyformed tissue with electrical stimulation to cause the newly formedtissue to contract in synchrony with the heart to improve the cardiacfunction.

The repopulation of the damaged myocardium with undifferentiatedcontractile cells can be carried out using a cellular or a molecularapproach. Cellular approaches involve the injection, either directly orvia coronary infusion, for example, of undifferentiated contractilecells, preferably cultured autologous skeletal cells, into the infarctzone (i.e., the damaged region of the myocardium). Molecular approachesinvolve the injection, either directly or via coronary infusion, forexample, of nucleic acid, whether in the form of naked, plasmid DNA,optionally incorporated into liposomes or other such delivery vehicle,or a genetically engineered vector into the infarct zone to convertfibroblasts, for example, invading the infarct zone into myoblasts. Thegenetically engineered vector can include a viral expression vector suchas a retrovirus, adenovirus, or an adeno-associated viral vector, forexample.

Various embodiments of the present invention provide one or more of thefollowing advantages: restoration of elasticity and contractility to thetissue; increased left ventricular function; reduction in the amount ofremodeling (i.e., conversion of elastic and contractile tissue toinelastic and noncontractile tissue); and decreased morbidity andmortality.

In one embodiment, the present invention provides an implantable systemcomprising: a cell repopulation source comprising genetic material,undifferentiated contractile cells, or a combination thereof capable offorming new contractile tissue in and/or near an infarct zone of apatient's myocardium; and an electrical stimulation device forelectrically stimulating the new contractile tissue in and/or near theinfarct zone of the patient's myocardium. An infarct zone of amyocardium can be determined by one of skill in the art. Near theinfarct zone means sufficiently close that damage to necrotic heartmuscle is realized. Typically, this means within about 1 centimeter (cm)of the edge of the infarct zone.

In another embodiment, the present invention provides an implantablesystem comprising: a cell repopulation source comprising skeletal musclesatellite cells capable of forming new contractile tissue in and/or nearan infarct zone of a patient's myocardium; and an electrical stimulationdevice for electrically stimulating the new contractile tissue in and/ornear the infarct zone of the patient's myocardium, wherein theelectrical stimulation device provides burst stimulation.

The present invention also provides a method of repairing the myocardiumof a patient, the method comprising: providing an implantable system asdescribed above; implanting the cell repopulation source into and/ornear the infarct zone of the myocardium of a patient; allowingsufficient time for the new contractile tissue to form from the cellrepopulation source; and electrically stimulating the new contractiletissue. Typically, new contractile tissue forms within about 15 days,although electrical stimulation may not be effective for up to about 14additional days after the contractile tissue forms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of an implantable system according to thepresent invention.

FIG. 2 is an illustration of a series of pulses a preferred electricalstimulation device provides during ventricular contractions.

FIG. 3 is a block diagram illustrating various components of animplantable pulse generator (IPG) that can be used according to methodsof the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods and implantable systems thatreverse the damage to necrotic heart muscle following myocardialinfarction by repopulating the damaged myocardium with undifferentiatedcontractile cells. This repopulation is augmented with electricalstimulation to assure synchrony of the contraction of the newly infusedtissue with cardiac contraction.

The repopulation of the damaged myocardium with undifferentiatedcontractile cells can be carried out using a variety of cellular ormolecular approaches. Typically, any of a variety of techniques by whichundifferentiated contractile cells repopulate the infarct zone of themyocardium can be used. They can involve delivering undifferentiatedcontractile cells to the infart zone or transforming cells and growingundifferentiated contractile cells in situ, for example.

Cellular approaches involve the injection, either directly or viacoronary infusion, for example, of undifferentiated contractile cells,preferably cultured myoblasts (i.e., muscle cells), and more preferably,skeletal or cardiac myoblasts into the infarct zone (i.e., the damagedregion of the myocardium) of the heart. Preferably, the cells areautologous to reduce and/or eliminate the immune response and tissuerejection. Typically, upon injection, skeletal myoblasts differentiateinto cardiac muscle fibers.

Molecular approaches involve the injection, either directly or viacoronary infusion, for example, of nucleic acid, whether in the form ofnaked, plasmid DNA, optionally incorporated into liposomes or similarvehicle, or a genetically engineered vector, into the infarct zone toconvert blastular, undifferentiated cells (e.g., fibroblasts or stemcells) invading the infarct zone into myoblasts. The vector can be aviral vector, preferably, an adenoviral vector, that expresses myogeninor MyoD, for example, which are members of the muscle family gene whosegene products induce fibroblast to myoblast phenotypic conversion.

These regions of repopulated cells provide improved diastolic cardiacfunction. Significantly, augmenting the repopulated regions withelectrical stimulation provides improved systolic as well as diastolicfunction. As a result, the present invention provides systems andmethods that include a cell repopulation source (i.e., a cellrepopulating agent) and an electrical stimulation device (i.e. astimulation source). The cell repopulation source can includeundifferentiated contractile cells such as autologous muscle cells, ornucleic acid for conversion of fibroblasts, for example, to myoblasts.The cell repopulation source can be delivered by direct injection intothe myocardium or via the coronary vasculature. Cell repopulation can becarried out using a syringe, or alternatively, a delivery device such asa catheter can be used. The cells or genetic material can be deliveredsimultaneously with the electrical stimulation device, or they can bedelivered separately. Preferably, the electrical stimulation device isthe carrier of the cells or genetic material. The electrical stimulationdevice typically includes an implantable muscle stimulator andelectrodes. Significantly, it does not include leads connecting it toany other device.

The cell repopulation source (i.e., cell repopulating agent) can includemedicaments, enhancing chemicals, proteins, and the like, forstimulating local angiogenesis, cell contractility, cell growth, andmigration, for example. These can include, for example, aFGF (acidicfibroblast growth factor), VEGF (vascular endothelial growth factors),tPA (tissue plasminogen activator), BARK (β-adrenergic receptor kinase),β-blockers, etc. Heparin, or other anticoagulants, such as polyethyleneoxide, hirudin, and tissue plasminogen activator, can also beincorporated into the cell repoputation source prior to implantation inan amount effective to prevent or limit thrombosis.

Referring to FIG. 1, an implantable system of the present inventioninclude a delivery device 10 comprising a carrier 22 forundifferentiated contractile cells and/or genetic material (i.e.,nucleic acid in a variety of forms), which is in the form of anelectrical stimulator capsule. If desired, other carriers can bedesigned depending on whether direct injection or coronary infusion isused. As shown in FIG. 1, the carrier 22 is delivered to the infarctzone of a patient's myocardium using a catheter 19. Optionally, nocarrier is required for delivery of the cells and/or genetic material,as when the cells and/or genetic material are systemically injected. InFIG. 1, the cell repopulation source is a fibroblast to myoblastconversion vector 14. The cell repopulation source (i.e., cellrepopulating agent) is typically released from the carrier 22 by passivediffusion into the infarct zone 16 of a myocardium 18 of a patient'sheart.

Undifferentiated Contractile Cells

Cells suitable for implantation in the present invention include a widevariety of undifferentiated contractile cells. Typically, thesedifferentiate to form muscle cells, however, they can be fibroblaststhat have been converted to myoblasts ex vivo, or any of a wide varietyof immunologically neutral cells that have been programmed to functionas undifferentiated contractile cells. Suitable cells for use in thepresent invention typically include umbilical cells, skeletal musclesatellite cells, and cardiac cells, whether they are autologous orallogeneic, genetically engineered or nonengineered. Mixtures of suchcells can also be used. Autologous cells are particularly desirable. Thecells are capable of repopulating the infarct zone of the myocardium.

Skeletal muscle satellite cells are particularly suitable for use in thepresent invention because they can differentiate to muscle cells thatare capable of contracting in response to electrical stimulation. Theyare also particularly suitable for use in the present invention becausethey can be obtained from cell cultures derived from the biopsy samplesof the same patient. Biopsy samples contain mature skeletal fibers alongwith reserve cells surrounding the mature fibers. Once placed inculture, reserve cells proliferate and their numbers quickly increase.These newly cultured cells can be injected back into the heart in and/ornear the infarct zone. Once in the heart muscle, the skeletal myoblastsfuse to form multinucleated myotubes having contractile characteristics.

Although skeletal muscle cells are capable of contracting, they aredifferent than cardiac cells. The mechanical and electricalcharacteristics of skeletal muscle are quite different than those ofheart muscle. Skeletal muscle satellite cells mechanically contract andrelax very rapidly. Therefore, in order to generate sustainedcontractions, skeletal cells are pulsed fairly rapidly, but this causedquick deprivation of energy reserves and the development of musclefatigue. However, skeletal muscle can be conditioned to contract atrates similar to or in conjunction with heart muscle.

Skeletal cells also differ from cardiac cells in their electricalcharacteristics. Each skeletal muscle fiber is stimulated byacetylcholine released from the motor neuron innervating the muscle.However, cardiac cells are interconnected via interclated diskscontaining channels for the passage of ions between the cytoplasm of thecells. This type of electrical interconnection does not exist betweenskeletal muscle satellite cells. The use of electrical stimulationcircumvents this problem and conditions the cells to contract at ratessimilar to or in conjunction with heart muscle.

The undifferentiated contractile cells can be delivered in combinationwith a delivery vehicle, such as liposomes or a polymeric matrix, asdescribed in greater detail below.

Once the undifferentiated cells form contractile tissue, their functioncan be further enhanced by metabolically altering them, for example, byinhibiting the formation of myostatin. This increases the number ofmuscle fibers.

Genetic Material

Nucleic acid can be used in place of, or in addition to, theundifferentiated contractile cells. The nucleic acid can be in the formof naked, plasmid DNA, which may or may not be incorporated intoliposomes or other such vehicles, or vectors incorporating the desiredDNA. The nucleic acid is capable of converting noncontracting cellswithin and/or near the infarct zone of a patient's myocardium tocontracting (i.e., contractile) cells. If desired, however,nonundifferentiated contractile cells can be converted toundifferentiated contractile cells using ex vivo genetic engineeringtechniques and then delivered to the infarct zone.

There are a wide variety of methods that can be used to deliver nucleicacid to nonundifferentiated contractile cells, such as fibroblast cells,to convert their phenotype from connective to contractile. Such methodsare well known to one of skill in the art of genetic engineering. Forexample, the desired nucleic acid can be inserted into an appropriatedelivery vehicle, such as, for example, an expression plasmid, cosmid,YAC vector, and the like, to produce a recombinant nucleic acidmolecule. There are a number of viruses, live or inactive, includingrecombinant viruses, that can also be used. A retrovirus can begenetically modified to deliver any of a variety of genes. Adenoviruscan also be used to deliver nucleic acid capable of convertingnonundifferentiated contractile cells to undifferentiated contractilecells, preferably, muscle cells. A “recombinant nucleic acid molecule,”as used herein, is comprised of an isolated nucleotide sequence insertedinto a delivery vehicle. Regulatory elements, such as the promoter andpolyadenylation signal, are operably linked to the nucleotide sequenceas desired.

The nucleic acid molecules, preferably recombinant nucleic acidmolecules, can be prepared synthetically or, preferably, from isolatednucleic acid molecules, as described below. A nucleic acid is “isolated”when purified away from other cellular constituents, such as, forexample, other cellular nucleic acids or proteins, by standard techniqueknown to those of ordinary skill in the art. The coding region of thenucleic acid molecule can encode a full length gene product or afragment thereof, or a novel mutated or fusion sequence. The codingsequence can be a sequence endogenous to the target cell, or exogenousto the target cell. The promoter, with which the coding sequence isoperably associated, may or may not be one that normally is associatedwith the coding sequence.

Almost any delivery vehicle can be used for introducing nucleic acidsinto the cardiovascular system, including, for example, recombinantvectors, such as one based on adenovirus serotype 5, Ad5, as set forthin French, et al., Circulation, 90, 2414-2424 (1994). An additionalprotocol for adenovirus-mediated gene transfer to cardiac cells is setforth in WO 94/11506, Johns, J. Clin. Invest., 96, 1152-1158 (1995), andin Barr, et al., Gene Ther., 1, 51-58 (1994). Other recombinant vectorsinclude, for example, plasmid DNA vectors, such as one derived frompGEM3 or pBR322, as set forth in Acsadi, et al., The New Biol., 3,71-81, (1991), and Gal, etal., Lab. Invest., 68, 18-25 (1993),cDNA-containing liposomes, artificial viruses, nanoparticles, and thelike.

The regulatory elements of the recombinant nucleic acid molecules arecapable of directing expression in mammalian cells, specifically humancells. The regulatory elements include a promoter and a polyadenylationsignal. In addition, other elements, such as a Kozak region, may also beincluded in the recombinant nucleic acid molecule. Examples ofpolyadenylation signals useful to practice the present inventioninclude, but are not limited to, SV40 polyadenylation signals and LTRpolyadenylation signals. In particular, the SV40 polyadenylation signalwhich is in pCEP4 plasmid (Invitrogen, San Diego, Calif.), referred toas the SV40 polyadenylation signal, can be used.

The promoters useful in constructing the recombinant nucleic acidmolecules may be constitutive or inducible. A constitutive promoter isexpressed under all conditions of cell growth. Exemplary constitutivepromoters include the promoters for the following genes: hypoxanthinephosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase,β-actin, human myosin, human hemoglobin, human muscle creatine, andothers. In addition, many viral promoters function constitutively ineukaryotic cells, and include, but are not limited to, the early andlate promoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter,the long terminal repeats (LTRs) of Maloney leukemia virus, HumanImmunodeficiency Virus (HIV), Cytomegalovirus (CMV) immediate earlypromoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), and otherretroviruses, and the thymidine kinase promoter of herpes simplex virus.Other promoters are known to those of ordinary skill in the art.

Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promote(increase) transcription in the presence of certain metal ions. Otherinducible promoters are known to those of ordinary skill in the art.

Promoters and polyadenylation signals used are preferably functionalwithin the cells of the patient. In order to maximize proteinproduction, regulatory sequences may be selected which are well suitedfor gene expression in the cardiac cells into which the recombinantnucleic acid molecule is administered. For example, the promoter ispreferably a cardiac tissue-specific promoter-enhancer, such as, forexample, cardiac isoform troponin C (cTNC) promoter. Parmacek, et al.,J. Biol. Chem., 265, 15970-15976 (1990), and Parmacek, et al., Mol. CellBiol., 12, 1967-1976 (1992). In addition, codons may be selected whichare most efficiently transcribed in the cell. One having ordinary skillin the art can produce recombinant nucleic acid molecules which arefunctional in the cardiac cells.

Genetic material can be introduced into a cell or “contacted” by a cellby, for example, transfection or transduction procedures. Transfectionrefers to the acquisition by a cell of new genetic material byincorporation of added nucleic acid molecules. Transfection can occur byphysical or chemical methods. Many transfection techniques are known tothose of ordinary skill in the art including: calcium phosphate DNAco-precipitation; DEAE-dextran DNA transfection; electroporation; nakedplasmid adsorption, and cationic liposome-mediated transfection.Transduction refers to the process of transferring nucleic acid into acell using a DNA or RNA virus. Suitable viral vectors for use astransducing agents include, but are not limited to, retroviral vectors,adeno associated viral vectors, vaccinia viruses, and Semliki Forestvirus vectors.

Treatment of cells, or contacting cells, with recombinant nucleic acidmolecules can take place in vivo or ex vivo. For ex vivo treatment,cells are isolated from an animal (preferably a human), transformed(i.e., transduced or transfected in vitro) with a delivery vehiclecontaining a nucleic acid molecule encoding an ion channel protein, andthen administered to a recipient.

In one preferred embodiment of in vivo treatment, cells of an animal,preferably a mammal and most preferably a human, are transformed in vivowith a recombinant nucleic acid molecule of the invention. The in vivotreatment typically involves local internal treatment with a recombinantnucleic acid molecule. When performing in vivo administration of therecombinant nucleic acid molecule, the preferred delivery vehicles arebased on noncytopathic eukaryotic viruses in which nonessential orcomplementable genes have been replaced with the nucleic acid sequenceof interest. Such noncytopathic viruses include retroviruses, the lifecycle of which involves reverse transcription of genomic viral RNA intoDNA with subsequent proviral integration into host cellular DNA. Mostuseful are those retroviruses that are replication-deficient (i.e.,capable of directing synthesis of the desired proteins, but incapable ofmanufacturing an infectious particle). Such genetically alteredretroviral expression vectors have general utility for high-efficiencytransduction of genes in vivo. Standard protocols for producingreplication-deficient retroviruses (including the steps of incorporationof exogenous genetic material into a plasmid, transfection of apackaging cell line with plasmid, production of recombinant retrovirusesby the packaging cell line, collection of viral particles from tissueculture media, and infection of the target cells with viral particles)are well known to those of skill in the art.

A preferred virus for contacting cells in certain applications, such asin in vivo applications, is the adeno-associated virus, adouble-stranded DNA virus. The adeno-associated virus can be engineeredto be replication deficient and is capable of infecting a wide range ofcell types and species. It further has advantages such as heat and lipidsolvent stability, high transduction frequencies in cells of diverselineages, including hemopoietic cells, and lack of superinfectioninhibition thus allowing multiple series of transductions.

Exemplary nucleic acid that would function as nucleic acid forincorporation into the cells include, but are not limited to, nucleicacid operably encoding a myogenic protein or MyoD protein. The nucleicacid can include an entire gene or a portion of a gene. Exemplary genesinclude, but are not limited to, the active forms of the myogenin geneor the MyoD gene.

The gene sequence of the nucleic acid delivered by the delivery vehicle(preferably, virus), including nucleic acid encoding proteins,polypeptide or peptide is available from a variety of sources includingGenBank (Los Alamos National Laboratories, Los Alamos, N. Mex.), EMBLdatabases (Heidelberg, Germany), and the University of WisconsinBiotechnology Center, (Madison, Wis.), published journals, patents andpatent publications. All of these sources are resources readilyaccessible to those of ordinary skill in the art. The gene sequence canbe obtained from cells containing the nucleic acid fragment (generally,DNA) when a gene sequence is known. The nucleic acid can be obtainedeither by restriction endonuclease digestion and isolation of a genefragment, or by polymerase chain reaction (PCR) using oligonucleotidesas primers either to amplify cDNA copies of mRNA from cells expressingthe gene of interest or to amplify cDNA copies of a gene from geneexpression libraries that are commerically available. Oligonucleotidesor shorter DNA fragments can be prepared by known nucleic acid synthesistechniques and from commercial suppliers of custom oligonucleotides suchas Amitof Biotech Inc. (Boston, Mass.), or the like. Those skilled inthe art will recognize that there are a variety of commercial kitsavailable to obtain cDNA from mRNA (including, but not limited toStratagene, La Jolla, Calif. and Invitrogen, San Diego, Calif.).Similarly, there are a variety of commercial gene expression librariesavailable to those skilled in the art including libraries available formStratagene, and the like. General methods for cloning, polymerase chainreaction and vector assembly are available from Sambrook et al. eds.(Molecular Cloning: A Laboratory Manual, 1989 Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) and Innis, et al. eds. (PCRStrategies, 1995, Academic Press, New York, N.Y.).

Depending on the maximum genome size that a particular viral genome canaccommodate or that can be associated with a virus particle, the virusdelivering nucleic acid to the cell can include nucleic acid encodingone or more proteins, polypeptides, or peptides. Oligonucleotides can bedelivered by virus through the incorporation of oligonucleotides withinthe virus or associated with the outer surface of the virus usingmethods well known to one of skill in the art.

Delivery Vehicles and Carriers

In addition to viral vector delivery vehicles, the cell repopulatingagent, whether it be genetic material or undifferentiated contractilecells, can include liposomes or a polymeric matrix. These can be coatedon or otherwise incorporated into a carrier, which can be the electricalstimulation device.

The cells and/or genetic material can be delivered in liposomes, whichare spherical particles in an aqueous medium, formed by a lipid by layerenclosing an aqueous compartment. Liposomes for delivery of geneticmaterial, for example, are commercially available from ClontechLaboratories UK Ltd., Basingstoke, Hampshire, United Kingdom.

The cells and/or genetic material can be delivered in a polymeric matrixthat encapsulates them. The polymeric matrix of this invention can beprepared from a homopolymer, a copolymer (i.e., a polymer of two or moredifferent monomers), or a composition (e.g., a blend) comprising fibrin,for example, with one or more polymers or copolymers, for example. Thecomposition preferably forms a viscoelastic, tear-resistant,biocompatible polymer. The term “viscoelastic” refers to the prescribed“memory” characteristics of a molecule that allow the molecule torespond to stress as if the molecule was a combination of elastic solidsand viscous fluids. The term “tear resistent” indicates that when thepolymer is exposed to expansion stress, the material does notsubstantially tear. Tearing refers to the propagation of a nick or cutin the material while under stress. The term “biocompatible” is usedherein to refer to a material that does not have toxic or injuriouseffects on biological systems.

Preferably, the polymeric matrix minimizes or does not exacerbateirritation to the heart wall when the cells and genetic material are inposition. The polymeric matrix is preferably nonthrombogenic whenapplied alone or alternatively when used with anti-thrombogenic agentssuch as heparin, and the like, or with anti-inflammatory agents such asdexamethasone, and the like. The polymeric matrix can be a biostable ora bioabsorbable polymer depending on the desired rate of release or thedesired degree of polymer stability.

The polymeric matrix of this invention can include one or more othersynthetic or natural polymers. Suitable polymers include those that arecompatible with the cells or genetic material. They can be biostable orbiodegradable. These include, but are not limited to, fibrins,collagens, alginates, polyacrylic acids, polylactic acids, polyglycolicacids, celluloses, hyaluronic acids, polyurethanes, silicones,polycarbonates, and a wide variety of others typically disclosed asbeing useful in implantable medical devices. Preferably, the polymersare hydrophilic.

Preferably, when genetic material, such as a genetically engineeredvector, is delivered, it can be incorporated into a crosslinkedhydrophilic polyacrylic acid polymer. This would form a high molecularweight hydrogel that could be used as a coating on a carrier, such asthe electrical stimulation device. The genetic material is preferablyincorporated into the hydrogel just prior to delivery by first swellingthe hydrogel.

Preferably, when undifferentiated contractile cells are delivered, theycan be incorporated into a gel of type I collagen. The cells can beinitially incorporated into media that includes type I collagensolution. This material can then be poured into a mold containing acarrier, such as the electrical stimulation device. After incubation ata temperature (e.g., 37° C.) and for a time (e.g., 30 minutes)sufficient to crosslink collagen, the coated device can be removed. Ifneeded, the resultant gel/stimulator can be cultured in media for a time(e.g., 14 days) sufficient to allow for cell growth.

Depending on the time of cell integration and proliferation, thepolymeric matrix can be in the form of a porous scaffold. This can bemade out of polyurethane using a dissolvable salt, as is known in theart of coating stents. The porous polymeric matrix can be coated withextracellular matrix components, such as fibronectin, heparin sulfate,etc., and then seeded with the undifferentiated contractile cells. Thecells can then grow out of the scaffold.

If desired, a fibrin matrix can be used. It can be prepared, forexample, by use of a fibrinogen solution and a solution of afibrinogen-coagulating protein. Fibrin is clotted by contactingfibrinogen with a fibrinogen-coagulating protein such as thrombin. Thefibrinogen is preferably used in solution with a concentration of about10 to about 50 mg/ml with a pH of about 5.89 to about 9.0 and with anionic strength of about 0.05 to about 0.45. The fibrinogen solutiontypically contains proteins and enzymes such as albumin, fibronectin,Factor XIII, plasminogen, antiplasmin, and Antithrombin III. Thethrombin solution added to make the fibrin is typically at aconcentration of up to about 120 NIH units/ml with a preferredconcentration of calcium ions between about 0.02 M and 0.2 M. Alsopreferably, the fibrinogen and thrombin used to make fibrin in thepresent invention are from the same animal or human species as that inwhich the cells or genetic material of the present invention will beimplanted to avoid cross-species immune reactions. The resulting fibrincan also be subjected to heat treatment at about 150° C., for about 2hours to reduce or eliminate antigenicity.

The optional carrier for delivery of the cells and/or genetic materialcan include the electrical stimulation device, for example, if the cellsand/or genetic material are directly injected into the infarct zone ofthe myocardium. Alternatively, the carrier for delivery of the cellsand/or genetic material can include catheters, for example, if the cellsand/or genetic material are to be injected via coronary infusion.

The cells and/or genetic material can be associated with the carrier asa coating or a preformed film, for example. If desired, the carrier canbe initially coated with an adhesive, such as that available under thetrade name CELLTAK BIOCOAT Cell Environments available from StratechScientific Ltd., Luton, Bedfordshire, United Kingdom, to enhanceadhesion of the polymeric matrix containing the undifferentiatedcontractile cells and/or genetic material.

The genetic material and/or undifferentiated contractile cells can alsobe delivered in a pharmaceutical composition using a catheter, forexample. Such pharmaceutical compositions can include, for example, thenucleic acid, in the desired form, and/or cells in a volume ofphosphate-buffered saline with 5% sucrose. In other embodiments of theinvention, the nucleic acid molecule and/or cells are delivered withsuitable pharmaceutical carriers, such as those described in the mostrecent edition of Remington's Pharmaceutical Sciences, A. Osol, astandard reference text in this field.

If genetic material and/or undifferentiated contractile cells areinjected into a patient separately from the electrical stimulationdevice, and are in fluid form, a catheter is advanced to the desiredsite for treatment, e.g., adjacent the site where the electricalstimulation device is to be positioned. The outer distal end of thecatheter is open or porous, thus permitting genetic material and/orundifferentiated contractile cells in fluid form to be dispensed out ofthe end. A reservoir connected to the catheter holds a supply of theselected genetic material and/or undifferentiated contractile cells.Control elements are used for adjustment of the pressure and flow rate,and may be mechanically or electronically controlled. Reference is madeto International Publication No. WO 95/05781, for a more detaileddescription of such a reservoir and catheter combination. This deliverydevice may or may not include a pump, such as an osmotic pump, fordelivering the cell repopulation source.

Electrical Stimulation Devices

The present invention also includes an electrical stimulation device 22.This provides the necessary electrical pulses at the correct time tomake the newly formed contractile tissue beat in synchrony with the restof the heart muscle.

The electrical stimulation device can include a muscle stimulator andseparate electrodes. Alternatively, the electrodes can be incorporatedinto the muscle stimulator. Furthermore, the muscle stimulator should,of course, include a battery for providing electrical current toelectrical and electronic circuitry.

The electrical stimulation device can provide burst stimulation, whichis typically used for stimulating skeletal muscle cells, or it canprovide synchronous single pulse stimulation, which is typically usedfor stimulating cardiac muscle cells. Alternatively, the electricalstimulation device can provide both burst and synchronous single pulsestimulation. This is particularly desirable if the new contractiletissue formed includes both skeletal and cardiac muscle cells and/orskeletal muscle cells are initially formed and then converted to cardiacmuscle cells. A pressure lead, or other means of monitoring aphysiological condition such as wall acceleration or intraventricularpressure, can be used to determine when to switch from burst mode tosingle phase mode of stimulation. If desired, two electrical stimulationdevices can be used, one that provides burst stimulation and one thatprovides synchronous single pulse stimulation.

Thus, conventional implantable pulse generators (IPGs) may be modifiedin accordance with the teachings of the present invention to provide anelectrical stimulation device 22, although they are typically notdesirable to stimulate cardiac muscle tissue that has been infused withcells and/or genetic material because the newly formed contractiletissue typically requires a burst stimulation to create a long-sustainedcontraction. Preferred systems of the present invention include animplantable stimulator (22 in FIG. 1) and two electrodes (cathode 30 andanode 32 in FIG. 1). Such a stimulator 22 may not include physical leadsconnecting it to any other device. However, it is possible to provideelectrical stimulation from a stimulator implanted in the body at aremote site and connected to the infarct zone using leads. Although thiswill make the clinical implementation more invasive in nature, it wouldreduce the complications of the stimulator capsule.

The preferred stimulator 22 shown in FIG. 1 is in the form of a capsulehaving electrodes 30 and 32 at either end. These electrodes provideelectrical contacts for the internal circuitry to sense the passage ofthe activation wavefront as well as to deliver a series of stimulationpulses necessary to cause the sustained contraction of the newly formedtissue, e.g., skeletal muscle tissue. The stimulator 22 also preferablyincludes two electronic circuits, a sense amplifier circuit, and a burstgenerator circuit.

The sense amplifier circuit is associated with a filter to form a senseamplifier. This is used to sense the electrical depolarization waveformas it passes through the infarct zone. While the amplifier increases thegain of this weak signal to the detection circuit, the filter helps toreject the noise signals from nearby muscles and any other electricaldevices.

The burst generator circuit provides a series of pulses as shown in FIG.2 during the ventricular contractions to keep newly formed skeletalmuscle tissue contracted. This is necessary to provide a sustainedcontraction during systole since the skeletal muscle relaxes much fasterthan cardiac muscle. The stimulator 22 also includes a power supply thatprovides electrical power to the sense amplifier and the burstgenerator.

The stimulator 22 can be in the shape of a cylinder, or otherappropriate shape suitable for implantation, and of a size sufficientlysmall for implantation. For example, it can be about 5 mm in diameterand 20 mm in length. Preferred materials include titanium, but otherbiocompatible materials can also be used. Stimulator 22 may contain abattery or other power source, electronics to detect heart beats andproduce burst stimulation, and telemetry circuits for triggeringstimulation on demand. Such circuitry can be developed by one of skillin the art, particulary in view of the teachings of U.S. Pat. No.5,697,884 (Francischelli et al.), U.S. Pat. No. 5,658,237(Francischelli), U.S. Pat. No. 5,207,218 (Carpentier et al.), U.S. Pat.No. 5,205,810 (Guiraudon et al.), U.S. Pat. No. 5,069,680 (Grandjean),and U.S. Pat. No. 4,411,268 (Cox).

Because a preferred stimulator 22 does not include physical leadsconnecting it to any other device, stimulator 22 needs to generate itsown electrical power. If the heart is assumed to be a 500Ω load, and 10pulses are needed at 10 volts for 1 millisecond, then each stimulationwill require 0.2 mJ. If the energy conversion has a 20% efficiency, thenthe 1 mJ of energy will be needed to stimulate the heart at every beat.Since the heart pumps about 50 mL of blood against 120 mm Hg (16 kPa),it does about 800 mJ of work. Therefore, the stimulator harvests aboutan-eighth of a percent of the mechanical work done by the heart. Thiscan be done by any of a variety of mechanisms that can converthydrostatic pressure to electrical energy.

Once implanted, typically, the stimulator 22 is preferably not activatedfor the first few weeks, to allow for contractile tissue growth. It isthen turned on with a low synchronization ratio (e.g., 3:1) between theintrinsic heart beats and the device-produced bursts and progressivelyincreased (e.g., to 1:1).

FIG. 3 is a block diagram illustrating various components of astimulator 22 which is programmable by means of an external programmingunit (not shown). One such programmer adaptable for the purposes of thepresent invention is the commercially available Medtronic Model 9790programmer. The programmer is a microprocessor device which provides aseries of encoded signals to stimulator 22 by means of a programminghead which transmits radio frequency encoded signals to IPG 51 accordingto a telemetry system, such as that described in U.S. Pat. No. 5,312,453(Wyborny et al.), for example.

Stimulator 22, illustratively shown in FIG. 3, is electrically coupledto the patient's heart 56 by lead 54. Lead 54, which includes twoconductors, is coupled to a node 62 in the circuitry of stimulator 22through input capacitor 60. In the presently disclosed embodiment, anactivity sensor 63 provides a sensor output to a processing/amplifyingactivity circuit 65 of input/output circuit 68. Input/output circuit 68also contains circuits for interfacing with heart 56, antenna 66, andcircuit 74 for application of stimulating pulses to heart 56 to moderatecontrol of software-implemented algorithms in microcomputer unit 78.

Microcomputer unit 78 comprises on-board circuit 80 which includessystem clock 82, microprocessor 83, and on-board RAM 84 and ROM 86. Inthis illustrative embodiment, off-board circuit 88 comprises a RAM/ROMunit. On-board circuit 80 and off-board circuit 88 are each coupled by adata communication bus 90 to digital controller/timer circuit 92. Theelectrical components shown in FIG. 3 are powered by an appropriateimplantable battery power source 94 in accordance with common practicein the art. For purposes of clarity, the coupling of battery power tothe various components of stimulator 22 is not shown in the figures.

Antenna 66 is connected to input/output circuit 68 to permituplink/downlink telemetry through RF transmitter and receiver unit 55.Unit 55 may correspond to the telemetry and program logic disclosed inU.S. Pat. No. 4,556,063 (Thompson et al.), or to that disclosed in theabove-referenced Wyborny et al. patent. Voltage reference (V_(REF)) andbias circuit 61 generates a stable voltage reference and bias currentfor the analog circuits of input/output circuit 68. Analog-to-digitalconverter (ADC) and multiplexer unit 58 digitizes analog signals andvoltages to provide “real-time” telemetry intracardiac signals andbattery end-of-life (EOL) replacement functions.

Operating commands for controlling the timing of stimulator 22 arecoupled by data bus 90 to digital controller/timer circuit 92, wheredigital timers and counters establish the overall escape interval of theIPG as well as various refractory, blinking, and other timing windowsfor controlling the operation of the peripheral components disposedwithin input/output circuit 68. Digital controller/timer circuit 92 ispreferably coupled to sensing circuitry 52, including sense amplifier53, peak sense and threshold measurement unit 57, andcomparator/threshold detector 59.

Sense amplifier 53 amplifies sensed electrocardiac signals and providesan amplified signal to peak sense and threshold measurement circuitry57. Circuitry 57, in turn, provides an indication of peak sensedvoltages and measured sense amplifier threshold voltages on path 64 todigital controller/timer circuit 92. An amplified sense amplifier signalis then provided to comparator/threshold detector 59. Sense amplifier 53may correspond to that disclosed in U.S. Pat. No. 4,379,459 (Stein).

Circuit 92 is further preferably coupled to electrogram (EGM) amplifier76 for receiving amplified and processed signals sensed by an electrodedisposed on lead 54. The electrogram signal provided by EGM amplifier 76is employed when the implanted device is being interrogated by anexternal programmer (not shown) to transmit by uplink telemetry arepresentation of an analog electrogram of the patient's electricalheart activity. Such functionality is, for example, shown in previouslyreferenced U.S. Pat. No. 4,556,063.

Output pulse generator 74 provides electrical stimuli to the patient'sheart 56 or other appropriate location through coupling capacitor 65 inresponse to a stimulation trigger signal provided by digitalcontroller/timer circuit 92. Output amplifier 74, for example, maycorrespond generally to the output amplifier disclosed in U.S. Pat. No.4,476,868 (Thompson).

It is understood that FIG. 3 is an illustration of an exemplary type ofdevice which may find application in the present invention, or which canbe modified for use in the present invention by one of skill in the art,and is not intended to limit the scope of the present invention.

Delivery Methods and Devices

The undifferentiated contractile cells and/or genetic material describedabove can be delivered into the infarct zone of the myocardium using avariety of methods. Preferably, the undifferentiated contractile cellsand/or genetic material are directly injected into the desired region.

For direct injection, a small bolus of selected genetic material and/orundifferentiated contractile cells can be loaded into a micro-syringe,e.g., a 100 μL Hamilton syringe, and applied directly from the outsideof the heart.

Preferably, however, the method of the present invention uses a catheterfor direct injection of both the electrical stimulation device and thecell repopulation source. For example, a catheter can be introduced fromthe femoral artery and steered into the left ventricle, which can beconfirmed by fluoroscopy. Alternatively, the catheter can be steeredinto the right ventricle.

The catheter includes an elongated catheter body, suitably an insulativeouter sheath which may be made of polyurethane, polytetrafluoroethylene,silicone, or any other acceptable biocompatible polymer, and a standardlumen extending therethrough for the length thereof, which communicatesthrough to a hollow needle element. The catheter may be guided to theindicated location by being passed down a steerable or guidable catheterhaving an accommodating lumen, for example as disclosed in U.S. Pat. No.5,030,204 (Badger et al.); or by means of a fixed configuration guidecatheter such as illustrated in U.S. Pat. No. 5,104,393 (Isner et al.).Alternately, the catheter may be advanced to the desired location withinthe heart by means of a deflectable stylet, as disclosed in PCT PatentApplication WO 93/04724, published Mar. 18, 1993, or by a deflectableguide wire as disclosed in U.S. Pat. No. 5,060,660 (Gambale et al.). Inyet another embodiment, the needle element may be ordinarily retractedwithin a sheath at the time of guiding the catheter into the patient'sheart.

Once in the left (or right) ventricle, the tip of the catheter can bemoved around the left ventricular wall as a prove to measure theelectrogram and to determine the location and extent of the infarctzone. This is a procedure known to one of skill in the art. Once theinfarct zone is identified, the steering guide will be pulled outleaving the sheath at the site of infarction. The cell repopulationsource and/or electrical stimulation device can then be sent down thelumen of the catheter and pushed into the myocardium. The catheter canthen be retracted from the patient.

The electrical stimulation device can include a variety of mechanismsfor holding it in place in the myocardium. For example, it can includeextendable hooks or talons. Alternatively, the tissue contacting portionof the device can be treated to achieve a microsurface texture (asdisclosed by Andreas F. von Recumin in: Biomaterials, 12, 385-389,“Texturing of Polymer Surfaces at the Cellular Level” (1991);Biomaterials, 13, 1059-1069, “Macrophage Response to MicrotexturedSilicone” (1992); and Journal of Biomedical Materials Research, 27,1553-1557, “Fibroblast Anchorage to Microtextured Surfaces” (1993)). Inan alternative embodiment, the stimulator can be in the form of a screwthat is driven into the muscle wall by turning.

EXAMPLES

The following examples are intended for illustration purposes only.

Example 1

Transformation of Fibroblasts in situ and Electrical Stimulation

Adenovirus expressing myogenin (Myogen adenovirus/cDNA, which can beproduced according to the method described by Murry et al., J. Clin.Invest., 98, 2209-2217 (1196)) was injected directly to the myocardiumusing a 100 microliter syringe. 10⁹ pfu (pfu-plaque forming units-onepfu is approximately 50 adenovirus particles) were diluted with salineto form a 100 microliter solution. This solution was kept on dry iceuntil the injection, and delivered in four equal amounts to theperimeter of the infarct zone, 90 degrees apart.

A histopathological assessment of the treated tissue was done to assessthe extent of fibroblast transformation. Tissue was processed forhistology and stained with H&E and Masson's Trichrome according tostandard methods.

Immunohistochemical staining was also done to determine whether therewas myogenin expression in the treated tissue. Eight μm frozen sectionswere cut from the tissue, fixed, and incubated with a rabbit polyclonalIgG that was raised against rat myogenin (Santa Cruz Biotechnology, Inc.Cat. No. sc-576). The samples were rinsed, incubated with a labeledsecondary antibody and visualized by epiflourescent microscopy.

Delivery of adenovirus expressing myogenin to infarcted tissue in vivoresulted in the appearance of multiple small patches of skeletalmyoblasts. These isolated muscle cells had peripheral nuclei, indicatingthat they were more likely to be skeletal muscle cells than cardiacmuscle cells as analyzed histologically. Typically, geneticallyconverted cells represented a more immature form of skeletal muscle thanthe myotubes seen in myoblast injected tissues (i.e., prior to fusion).No myogenin immunoreactivity was present in these cells at the time ofsacrifice. Therefore, it was concluded that the myogenin created by theadenovirus was no longer present at the time of the tissue harvest (aswas expected at two weeks after delivery of the virus, since adenovirusexpression does not pursue for more than one week to 10 days in vivo).

In cryoablated, adenovirus beta-galactosidase injected hearts, onlyfibroblasts and lymphocytic infiltrate along with small capillaries weredetected within the infarct, similar to the results obtained withanimals receiving cryoablation, but no viral injections (control).Thefore, in this control group, no muscle cells or positive staining formyogenin was detected. This comprised the placebo group of the moleculararm of the study.

Example 2

Injection of Contractile Cells and Electrical Stimulation in Canines

Growth and Passage Information for Skeletal Myoblast Cells

1. Growth Medium Formulation:

81.6% M199 (Sigma, M-4530)

7.4% MEM (Sigma, M4655)

10% Fetal Bovine Serum (Hyclone, Cat.# A-1115-L)

1× (1%) Penicillin/Streptomycin (Final Conc. 100,000 U/L Pen./10 mg/LStrep., Sigma, P-0781).

2. Cell Passage Information:

A. Seeding densities of 1×10⁴ cells/cm² will yield an 80% confluentmonolayer in approximately 96 hours.

B. Split ratios of 1:4-1:6 will yield a confluent monolayer within 96hours.

C. Do not allow the cells to become confluent. Cell to cell contact willcause the cells to differentiate into myotubes.

3. Passage Information:

ml of Trypsin Flask Size ml of HBSS Solution ml of Media/Flask T-25 3 310 T-75 5 5 20-35 T-150 10-15 10-15 40-60 T-225 15-25 15-25  60-125

A. Remove culture medium from T-flask.

B. Add back the appropriate amount of Hank's Balanced Salt Solution(HBSS).

C. Incubate for approximately 5 minutes at room temperature.

D. Remove HBSS and replace with the Trypsin solution.

E. Incubate for a maximum of 5 minutes at 37° C. in a 5% CO₂ incubator.Cells will detach from the cell culture substrate prior to 5 minutes.

F. Do not trypsinize for a longer period than necessary. The cells willbe shocked if allowed to remain in the trypsin for longer than 5minutes.

G. Gently agitate flask to remove cells.

H. Add back at least an equal volume of growth medium to neutralize thetrypsin.

I. Remove a sample for cell count.

J. Centrifuge the cells at 1000 RPM for 10 minutes.

K. Count cells and calculate cell numbers.

L. Resuspend in cell culture medium and seed into appropriate flasks.

M. To maintain a healthy culture, change medium every 2-3 days.

4. Cell Count:

A. Dilute cells into the appropriate diluent (Trypan Blue or HBSS). Nodilution or 1:2 dilution works well for a confluent T-flask.

B. Count cells using a hemocytometer. The most accurate range for thehemocytometer is between 20-50 cells/square.

C. Calculation:

Cells counted (divided by) squares counted (multiplied by) dilutionfactor (multiplied by) 1×10⁴=cells/ml in the original cell suspension.

Enzymatic Myoblast Isolation

Skeletal muscle, unlike cardiac muscle, retains the ability to repairitself if damaged. The reason for this is the presence ofundifferentiated myoblasts (also referred to as satellite cells) locatedin the mature muscle.

Mature muscle myotubes can't be grown in culture, because in the processof differentiating from myoblasts to myotubes the cells loose theability to proliferate. In order to conduct in vitro research onskeletal muscle myotubes it is first necessary to first isolate themuscle myoblasts. The following procedure is for isolating primarymuscle myoblasts from skeletal muscle biopsies and sub-culturing theresulting cells.

1. Materials:

A. Isolation Medium: 80.6% M199 (Sigma, M4530), 7.4% MEM (Sigma, M4655),10% Fetal Bovine Serum (Hyclone, Cat.# A-1115-L), 2× (2%)Penicillin/Streptomycin (Final Conc. 200,000 U/L Pen./20 mg/L Strep.,Sigma, P-0781).

B. Myoblast Growth Medium: 81.6% M199 (Sigma, M4530), 7.4% MEM (Sigma,M4655), 10% Fetal Bovine Serum (Hyclone, Cat.# A-1115-L), 1× (1%)Penicillin/Streptomycin (Final Conc. 100,000 U/L Pen./10 mg/L Strep.,Sigma, P-0781).

C. Wash Solution: M199, 2× Penicillin/Streptomycin.

D. Collagenase (Crude: Type IA, Sigma, C-2674).

E. Hyaluronidase (Type I-S, Sigma, H-3506).

F. Protease, from Streptomyces griseus, (Sigma, P-8811).

G. Hank's Balanced Salt Solution (HBSS), Ca²⁺ and Mg²⁺ free (Sigma,H-6648).

H. 70% EtOH (sterile filtered).

I. Percoll (Sigma, P-4937).

J. 0.5 g/L Trypsin Solution (Sigma, T-3924).

K. 15 ml and 50 ml Sterile Centrifuge Tubes.

L. 100 mm Sterile Petri Dish.

M . Sterile Scissors and Sterile Forceps (Fine Scientific Tools).

N. 5 ml, 10 ml, 25 ml Sterile Pipettes (Falcon).

O. BIOCOAT Laminin Cellware (25 cm² and 75 cm² flasks, Becton Dickinson,Cat. No(s). 40533, 40522)

P. T-75 Tissue Culture Flasks, 0.22 μm vented cap (Corning).

Q. Filter, 0.22 μm and 0.45 μm, cellulose acetate (Corning).

R. Polycarbonate Centrifuge Tubes.

S. Beckman Centrifuge, GS-6.

T. Incubator Shaker.

2. Method:

All steps of this procedure should be performed aseptically.

A. Prepare Isolation Medium:

Add approximately 30 ml to a 50 ml sterile centrifuge tube (10 gm biopsyor less).

Add approximately 50 ml to a 125 ml sterile media bottle (up to 25 gmbiopsy).

B. Place the Isolation Medium on ice or ice packs to keep cold(approximately 4° C.).

C. Prepare the enzyme solution, the same day it will be used; by adding1.0 gm collagenase and 0.2 gm hyaluronidase to 100 ml of M199 (100 ml ofenzyme/disbursing solution is enough to digest 40-50 gm of skeletalmuscle).

D. Filter sterilize the enzyme solution first through a 0.45 μm filterand then a 0.22 μm filter and keep at 4° C. until ready to use.

E. Prepare the disbursing solution, the same day it will be used, byadding 1 gm of the protease to 100 ml of M199.

F. Filter sterilize through a 0.22 μm filter and keep at 4° C. untilready to use.

G. Under semi-sterile conditions remove the skeletal muscle biopsy,preferably from the belly of the muscle, and place it into the isolationmedium.

H. Seal the container and store at approximately 4° C. until ready tomince.

I. Remove the tissue and place into a sterile petri dish.

J. Trim off any connective tissue and measure the final weight.

K. Rinse the tissue with sterile 70% EtOH for 30 seconds.

L. Aspirate the EtOH and rinse the tissue 2× with HBSS.

M. Finely mince the biopsy using scissors and tweezers.

N. Transfer the minced biopsy into 50 ml sterile centrifuge tubes. Nomore than 20 gm/tube to allow for effective enzymatic digestion.

O. Rinse the tissue by adding approximately 25 ml/tube of HBSS, mix, andpellet the tissue by centrifuging at 2000 RPM (allow the centrifuge toreach 2000 RPM and turn off).

P. Decant off the HBSS and repeat the rinse and centrifuge an additionaltwo more times.

Q. Add enzyme solution to the tubes (approximately 25 ml/15 gm-20 gmoriginal biopsy).

R. Incubate tubes in the incubator shaker for 20 minutes (Set Point—37°C., 300 RPM).

S. Centrifuge at 2000 RPM for 5 minutes and discard the supernatant.

T. Add disbursing solution to the tubes (approximately 25 ml/15 gm-20 gmoriginal biopsy).

U. Incubate tubes in the incubator shaker for 15 minutes (Set Point—37°C., 300 RPM).

V. Centrifuge at 2000 RPM for 5 minutes.

W. Harvest the supernatant, inactivate the enzyme by adding FBS to afinal concentration of 10%, and store at 4° C.

X. Add disbursing solution to the tubes for a second enzymatic digestion(approximately 25 ml/5 gm-20 gm original biopsy).

Y. Incubate tubes in the incubator shaker for 15 minutes (Set Point—37°C., 300 RPM).

Z. Centrifuge at 2000 RPM for 5 minutes.

AA. Harvest the supernatant and inactivate the enzyme by adding FBS to afinal concentration of 10%.

BB. Centrifuge the cell slurry from the disbursing digestion steps(refer to W and M) at 2400 RPM for 10 minutes.

CC. Remove and discard the supernatant.

DD. Resuspend the cell pellets in a minimal volume of Wash Solution.

EE. Combine the pellets in a 50 ml centrifuge tube, bring the volume upto 40 ml using Wash Solution.

FF. Centrifuge at 2400 RPM for 10 minutes.

GG. Remove the supernatant and repeat the cell wash two more times.

HH. On the final rinse resuspend the pellet in 2 ml of MEM. If theinitial biopsy was close to or greater than 25 gm resuspend into 4 ml ofMEM.

II. Prepare 20% Percoll and 60% Percoll in MEM.

JJ. Make the density gradient by layering 10 ml of 20% Percoll/MEM over5 ml of 60% Percoll/MEM (refer to FIG. 1).

KK. Add 2 ml of the cell suspension on the top of the 20% Percoll band.

LL. Use a scale to prepare a second tube as a counter balance forcentrifugation.

MM. Centrifuge at 11947 RPM (15000×g) for 5 minutes at 8° C. (adjustacceleration to 5 and brake to 0).

NN. Isolate the band of cells that develops between the 20% and 60%Percoll layers. This band contains the myoblast cells.

OO. Determine the volume of the band and dilute it with 5 volumes ofgrowth medium.

Note: If the Percoll isn't diluted with enough growth medium it will bevery difficult to pellet the myoblasts out of solution.

PP. Centrifuge at 3000 RPM for 10 minutes.

QQ. Remove the supernatant and resuspend the pellet in growth medium.

RR. Count the cells in suspension.

SS. Plate out the cells in the BIOCOAT Laminin coated T-flasks atapproximately 1×10⁴ cells/cm². The first plating should be done on alaminin coated surface to aid in cell attachment.

TT. Culture the cells to 60%-80% confluence. If the cells are allowed tobecome confluent they will terminally differentiate into myotubes.

UU. Trypsinization Procedure:

Wash the monolayer with HBSS

Add trypsin (0.5 g/l trypsin)

T-Flask ml of HBSS ml of Trypsin T-25 3 3 T-75 5 5 T-150 10 10

Incubate at 37° C. for no more than 5 minutes

Neutralize the trypsin with serum containing medium

Remove the cells

Centrifuge at 800 rpm for 10 minutes

Re-suspend in Myoblast Growth Medium

Seed cell culture treated T-flasks at approximately 1×10⁴ cells/cm².

Split ratio's of 1:4 to 1:6 work well for a 60-80% confluent culture.

Development of Canine Infarctmodel and Cell Injection

Myocardial infarction was created in the canine heart by cryoablating around region of the left ventricular free wall, approximately 3.5 cm indiameter. This was achieved by first performing a left thoracotomy atthe fourth or fifth intercostal space to have access to the canine'sheart. The heart was re-positioned to have access to the LV free wall. Aregion relatively free of coronary vasculature was identified forcryoablation.

The myocardium was infarcted by applying a custom cryoablationinstrument with a 3.5 cm diameter metal plate to the epicardial surfacefor up to 10 minutes. Since the probe was cooled with liquid nitrogen,its temperature was as cold as minus 180° C. before it was applied tothe surface of the heart. Because the volume of blood flowing within theleft ventricle of the dog is enough to warm the endocardial surface, atrue transmural ablation could not be achieved. Nevertheless, 13.5 gramsof the LV free wall, constituting 15 percent of the LV free wall mass,was ablated. This is a typical size of infarct for a human patient aswell.

For one dog, 1.79×10⁸ (one hundred and seventy nine million) myoblastswere obtained within 11 days from 2.5 grams of skeletal muscle biopsy.These cells were reinjected into the canine myocardium at ten locationsaround the ablation site, using a syringe with a 22 gauge needle andinjecting 0.5 mL per site (1.79×10⁸ cells/5 mL of saline).

In vivo Electrical Stimulation

Two weeks after the introduction of the MI, the chest was opened again,and the animal was instrumented as before. In addition, an electrode wasattached to the ventricular apex for unipolar VVI pacing. Two moreelectrodes were attached to either sides of the infarcted area tostimulate the cellular cardiomyoplasty region.

It was noticed at this time that the animal's LV pressures and strokevolume were not improved significantly. As a matter of fact, peaksystolic pressures were only slightly over 80 mm Hg, and the strokevolume was again around 22 mL when the animal was VVI paced. Thissuggests that cell placement alone did not appreciably improve thesystolic function.

When the skeletal muscle stimulator was turned on, systolic pressuresreached 100 mmHg, and stroke volumes increased to 40 mL. Due tosynchronization problems between the ventricular pacer and the skeletalmuscle stimulator, a stable trace could not be obtained during thestudy. Nevertheless, this experiment gave an indication that thepresence of the skeletal cells alone might not be enough to improve thesystolic function, and that there might be a need for skeletal musclestimulation to improve the cardiac function in conjunction with cellularcardiomyoplasty.

Changes in the wall motion in the region of treatment were also observedwith the application of skeletal muscle stimulation. With traditionalventricular pacing only (upper trace), the length of the infarct zoneshortened by only 0.5 mm. However, when skeletal muscle stimulation wasapplied in addition to ventricular pacing, the shortening about 1.0 mm,indicating that wall motion, or contractility, was increased byelectrically stimulating the skeletal cardiomyoplasty region.

Histopathological Methods and Results

In order to assure that the transplanted skeletal cells were present atthe end of the two week period, preserved tissue sections were analysedwith immuno-histochemistry using an anti-myosin to antibody (skeletal,fast, MY-32). Positive (green) staining at two different regions of theablated site indicated the presence of the injected skeletal musclecells in the ablated region of myocardium, two weeks after theirintroduction. This immuno-staining study provided definitive evidencefor the presence of skeletal muscle cells in the myocardium. Theimmuno-histochemistry staining protocol used is described as follows:

Immuno-histological staining protocol

Materials:

Monoclonal Anti-Skeletal Myosin (Fast), clone MY-32, Sigma, Cat.No.M4276.

Polyclonal Rabbit Anti-Connexin-43, Zymed, Cat.No. 71-0700.

Goat Anti-Mouse IgG-FITC, Sigma, Cat.No. F-0257.

Goat Anti-Rabbit IgG (Whole Molecule)-TRITC, Sigma, Cat.No. T-6778.

PBS, Sigma, Cat.No. 1000-3.

Goat Serum, Sigma.

Acetone, Sigma, Cat.No. A4206.

Mounting Medium, Sigma Cat.No. 10004.

Microscope, Nikon, Labophot-2.

Samples:

Skeletal Muscle (Control)

Posterior Lesion

Mid Lesion

Anterior Lesion

J (L) Ventricular Free Wall (Control)

Methods:

A. Clean glass slides with 95% EtOH and treat with poly-Lysine or buypre-treated slides.

B. Obtain tissue samples and freeze onto cryostat chucks.

C. Cut 8 μm thick cryostat sections of the frozen tissue block, place ontreated glass slides, and store at ≦−70° C.

D. Allow tissue sections to come to room temperature prior to initiatingstaining (approximately 15-30 minutes).

E. Fix samples in cold Acetone (≦−10° C.) for 10 minutes at 4° C.

F. Wash sample with PBS three times (care must be taken to avoid washingthe sample off of the slide).

G. Block samples with 10% Goat Serum/PBS for 20 minutes at roomtemperature, using a humidified chamber.

H. Dilute the first primary antibody, Connexin-43, 1:100 in PBScontaining 10% goat serum. Dilute enough antibody to cover the samples(approximately 150 μi), add to the tissue sections, and incubate in ahumidified chamber for 1 hour at room temperature.

I. Wash sample in 10% Goat Serum/PBS three times (5 minutes/wash).

J. Dilute the second primary antibody, My-32, 1:200 in PBS containing10% goat serum. Dilute enough antibody to cover the samples(approximately 150 μl), add to the tissue sections, and incubate in ahumidified chamber for 1 hour at room temperature

K. Wash samples in 10% Goat Serum/PBS three times (5 minutes/wash).

L. Dilute the secondary antibodies, mix the antibody solutions, and addto the tissue sections.

Anti-Rabbit IgG (Whole Molecule)-TRITC, 1:50 in PBS.

Anti-Mouse IgG-FITC, 1:100 in PBS.

M. Incubate in a dark, humidified chamber, for 45 minutes at roomtemperature.

N. Wash samples in PBS three times (5 minutes/wash).

O. Add mounting medium and a coverslip.

P. Read on the microscope using the FITC filter, the TRITC filter, andthe UV light source.

Q. Store samples in a dark chamber at ≦4° C.

The complete disclosures of the patents, patent applications, andpublications listed herein are incorporated by reference, as if eachwere individually incorporated by reference. The above examples anddisclosure are intended to be illustrative and not exhaustive. Theseexamples and description will suggest many variations and alternativesto one of ordinary skill in this art. All these alternatives andvariations are intended to be included within the scope of the attachedclaims. Those familiar with the art may recognize other equivalents tothe specific embodiments described herein which equivalents are alsointended to be encompassed by the claims attached hereto.

What is claimed is:
 1. An implantable system comprising: (a) a cellrepopulation source implanted into the myocardium comprising geneticmaterial, cultured undifferentiated contractile cells, or a combinationthereof, capable of forming new contractile tissue in and/or near aninfarct zone of a patient's myocardium; and (b) an electricalstimulation device for electrically stimulating a plurality ofcontractions of the new contractile tissue in and/or near the infarctzone of the patient's myocardium.
 2. The implantable system of claim 1wherein the electrical stimulation device provides burst stimulation. 3.The implantable system of claim 1 wherein the electrical stimulationdevice provides pulse stimulation.
 4. The implantable system of claim 1wherein the undifferentiated contractile cells comprise skeletal musclesatellite cells.
 5. The implantable system of claim 1 wherein theundifferentiated contractile cells comprise autologous cells.
 6. Theimplantable system of claim 1 wherein the genetic material comprises adelivery vehicle comprising a nucleic acid molecule.
 7. The implantablesystem of claim 6 wherein the nucleic acid molecule encodes a myogenicdetermination gene.
 8. The implantable system of claim 6 wherein thedelivery vehicle comprises a viral expression vector.
 9. The implantablesystem of claim 6 wherein the delivery vehicle comprises liposomes. 10.The implantable system of claim 1 wherein the genetic material comprisesnaked, plasmid DNA.
 11. The implantable system of claim 1 wherein thecell repopulation source further comprises a polymeric matrix.
 12. Theimplantable system of claim 1 wherein the cell repopulation source isassociated with a carrier.
 13. The implantable system of claim 12wherein the cell repopulation source is coated on a carrier.
 14. Theimplantable system of claim 1 wherein the electrical stimulation devicecomprises a muscle stimulator having two electrodes connected thereto.15. The implantable system of claim 14 wherein the muscle stimulator isimplantable and is in the form of a capsule having electrodesincorporated therein.
 16. The implantable system of claim 15 wherein themuscle stimulator is a carrier for the cell repopulation source.
 17. Animplantable system comprising: (a) a cell repopulation source comprisingskeletal muscle satellite cells capable of forming new contractiletissue in and/or near an infarct zone of a patient's myocardium; and (b)an electrical stimulation device for electrically stimulating aplurality of contractions of the new contractile tissue in and/or nearthe infarct zone of the patient's myocardium, wherein the electricalstimulation device provides burst stimulation.
 18. A method of repairingthe myocardium of a patient, the method comprising: (a) providing animplantable system comprising: (i) a cell repopulation source comprisinggenetic material, undifferentiated contractile cells, or a combinationthereof capable of forming new contractile tissue in and/or near aninfarct zone of a patient's myocardium; and (ii) an electricalstimulation device for electrically stimulating a plurality ofcontractions of the new contractile tissue in and/or near the infarctzone of the patient's myocardium; (b) implanting the cell repopulationsource into and/or near the infarct zone of the myocardium of a patient;(e) allowing sufficient time for new contractile tissue to form from thecell repopulation source; and (f) electrically stimulating the newcontractile tissue.
 19. The method of claim 18 wherein the electricalstimulation device comprises a muscle stimulator and electrodes whereinthe electrodes are implanted into and/or near the infarct zone of themyocardium.
 20. The method of claim 18 wherein the undifferentiatedcontractile cells comprise autologous cells.
 21. The method of claim 18wherein the muscle stimulator is implantable and is in the form of acapsule having electrodes incorporated therein.
 22. The method of claim21 wherein the muscle stimulator is a carrier for the cell repopulationsource.
 23. The method of claim 22 wherein the muscle stimulator andcell repopulation source are delivered to the infarct zone through acatheter.